The Role of the DnaX Complex in Assembling Pol III Initation Complexes

描述（由申请人提供）：所有细胞在细胞分裂过程中必须快速准确地复制其DNA。DNA复制是由复杂的多蛋白组装体进行的，这些组装体必须以高度协调的方式被募集并装载到模板DNA上。DNA聚合酶III（Pol III）全酶来自大肠杆菌E.大肠杆菌是研究DNA复制的模型系统。Pol III全酶的一个关键组分是5亚基DnaX复合物，其在将“滑动钳”持续合成因子加载到DNA上以及协调前导链和滞后链合成中具有已知的功能。DnaX亚基之一，DnaX基因的产物，在体内以全长或截短形式存在。虽然这两种形式都支持进行性DNA合成，但两种成分之间存在重要的功能差异。该提案的目标是确定DnaX蛋白的两种形式如何影响启动DNA复制的机制，从而更好地理解DnaX复合物的作用。将开发快速动力学测定来测量在DNA模板上组装Pol III全酶的速率。将使用KinTek快速混合/淬灭装置和荧光共振能量转移技术。将通过实验确定全长DnaX蛋白相对于截短形式的动力学优势。全酶/DNA起始复合物形成的机制将通过检查各全酶组分浓度变化时的动力学效应来阐述。这些实验将检验核心Pol III DNA聚合酶被含有全长DnaX蛋白的DnaX复合物“陪伴”到起始复合物的假设。遗传信息的快速准确复制是细胞分裂的基本过程。因此，加深我们对这一过程的理解的研究对于开发更有效的癌症或病原性感染治疗方法至关重要。这项提议的结果将确立DNA复制机制中一个关键组成部分的作用，并将导致对DNA复制如何在分子水平上启动的更深入理解。

DESCRIPTION (provided by applicant): All cells must rapidly and accurately replicate their DNA during the process of cell division. DNA replication is carried out by complex multi-protein assemblies, which must be recruited to and loaded onto the template DNA in a highly coordinated fashion. The DNA polymerase III (Pol III) holoenzyme from the bacterium E. coli is a model system for studies of DNA replication. A key component of the Pol III holoenzyme is the 5 subunit DnaX complex, which has known functions in loading the "sliding clamp" processivity factor onto the DNA and in coordinating leading and lagging strand synthesis. One of the DnaX subunits, the product of the DnaX gene, occurs in vivo as either a full length or truncated version. While both forms support processive DNA synthesis, there are important functional differences between the two components. The goal of this proposal is to ascertain how the two forms of the DnaX protein affect the mechanism for initiating DNA replication and to thereby better understand the role of the DnaX complex. Rapid kinetic assays will be developed to measure the rate for assembling the Pol III holoenzyme on a DNA template. Both a KinTek rapid mix/quench device and fluorescence resonance energy transfer techniques will be utilized. The kinetic advantage of the full length DnaX protein over the truncated version will be established experimentally. The mechanism by which holoenzyme/DNA initiation complexes are formed will be elaborated by examining the kinetic effects as the concentration of each holoenzyme component is varied. These experiments will test the hypothesis that the core Pol III DNA polymerase is "chaperoned" to the initiation complex by DnaX complexes containing the full length DnaX protein. PUBLIC HEALTH RELEVANCE The fast and accurate replication of genetic information is a fundamental process of cell division. Thus, research that deepens our understanding of this process is crucial for developing more effective treatments for cancer or pathogenic infection. The results of this proposal will establish the role of a key component in the DNA replication machinery and will lead to a greater understanding of how DNA replication is initiated at the molecular level.